Genetic engineering has made it possible to produce large amounts of heterologous proteins or polypeptides in bacterial cells by means of recombinant expression systems, especially by expression in such prokaryotes as Escherichia coli (E. coli).
The expressed heterologous proteins may be of mammalian, other eukaryotic, viral, bacterial, cyanobacterial, archaebacterial, or synthetic origin.
Unlike native bacterial proteins, which can often be efficiently accumulated within a bacterial cell even when encoded by a single chromosomal gene copy, there are no published reports to date of heterologous proteins being successfully accumulated within bacterial cells to levels exceeding 0.1% of total cell protein when expressed from a single chromosomal gene location.
0.1% of total cell protein (150 micrograms protein per trillion bacterial cells) is chosen as a practical measure of successful accumulation of protein because it approximately defines the lower limits of (a) economically significant accumulation of a desired protein by contemporary recombinant bacterial production standards, and (b) visual detection of a protein band by Coomassie-stained polyacrylamide gel analysis of whole bacterial cell extracts.
The relatively poor performance of non-bacterial genes when expressed in bacterial cells, even when placed under the control of the strongest known bacterial promoters, has been generally attributed to poor translation of the non-bacterial mRNAs and rapid degradation of newly synthesized non-bacterial proteins. It has almost universally been assumed that, in order to achieve successful accumulation of non-bacterial or heterologous proteins in bacterial cells, the genes encoding the heterologous proteins must be located on multicopy plasmid vectors.
A gene carried on one of the multicopy plasmids commonly used for cloning and expressing genes encoding heterologous proteins in E. coli usually has a copy number of more than 20 copies/cell. Even low copy number plasmids (e.g., pACYC177 and pLG339) generally exist at 6-10 copies per cell. One disadvantage imposed by plasmid gene dosages is that the expression of even minute amounts of some foreign proteins can kill host cells (see Meth. Enzymol. 185:63-65, ed. D. Goeddel, 1990). For this reason, it would be advantageous to reliably limit the copy number of genes encoding such toxic gene products, such as by integrating the gene into the bacterial chromosome at one or a small number of copies per cell. For example, such a system would allow one to make more representative cDNA expression libraries in bacterial hosts if the high-copy expression of one or more of the cDNAs in the library could kill the bacterial host or cause it to grow poorly.
Chromosomal integration of genes encoding heterologous polypeptides would also be advantageous as an alternative means for expression of heterologous proteins in bacterial host cells. Multicopy vectors are often unstable and require the use of antibiotics in the growth medium for maintenance. Present methods of integrating foreign genes into the bacterial chromosome suffer from inefficiency, the inability to control the site of integration of the foreign gene, and/or the inability to control the copy number of the integrated gene. Most importantly, all efforts to date to create recombinant DNA constructs on the bacterial chromosome, wherein a bacterial promoter is fused to a heterologous gene, have involved the creation of viral or plasmid intermediates carrying the construct. Because such intermediates replicate at high copy number, they may be difficult or even impossible to recover in cases where the foreign gene product is toxic to the bacterial cell. Expression of the encoded gene, even at low levels, may be toxic to the host cells, due to the high copy number of these intermediates, which effectively multiplies the level of expression.
Previous methods for achieving the integration of heterologous genes into the chromosome of a bacterial host include the use of phage lambda vectors. The phage DNA in circular form is inserted linearly into the bacterial chromosome by a single site specific recombination between a phage attachment site (attP), 240 bases long, and a bacterial attachment site (attB), only 25 bases long. The two sites have 15 bases in common. This site-specific recombination is catalyzed by a special integrase, specified by the phage gene INT (VIROLOGY pp. 56-57 (Lippincott, 2nd ed., R. Dulbecco and H. Ginsberg, eds., Philadelphia, Pa, 1985).
Phage vectors which are INT.sup.- can be integrated into the chromosome in a normal fashion as long as integrase is supplied in trans, e.g., by an INT+ helper phage (see, e.g., Borck et al. (1976) Molec. Gen. Genet. 146:199-207).
Phage vectors which are both att- and INT- can likewise be integrated into the bacterial chromosome as double lysogens by using att+INT+ helper phage. Double lysogens are formed by linkage of the prophages at the bacterial attachment site and are integrated into the chromosome by general bacterial recombination between homologous sequences on the defective phage and on the helper phage (see e.g., Struhl et al. (1976) Proc. Natl. Acad. Sci. USA 73:1471-1475). Similarly, it is also possible to integrate non-replicating colE1 replicons into the genome of polA strains of E. coli by means of recombination between the host chromosome and homologous sequences carried by the plasmid vector (Greener and Hill (1980) J. Bacteriol. 144:312-321).
More recently, systems have been specifically designed for the integration of foreign genes into a bacterial host chromosome. For example, U.S. Pat. No. 5,395,763 (Weinberg et al.) discloses a chromosomal expression vector for the expression of heterologous genes. This vector was created utilizing a multicopy number plasmid intermediate, into which the gene of interest is cloned, placing the gene in operable linkage with the bacteriophage middle promoter, Pm. This plasmid intermediate, which comprises a defective Mu genome (lacking the genes necessary for the formation of phage particles) is introduced into a packaging strain to produce infectious Mu particles, which are then used to introduce the vector into host cells and integrate the vector into the host cell genome. This vector system is amplifiable once integrated into the host cell genome, but the mechanism of amplification (replicative transposition) is normally toxic to the host cell, due to integration of the replicating prophage into essential host cell genes (Neidhardt et al., ESCHERICHIA COLI AND Society for Microbiology, Neidhardt et al. eds., Washington, D.C., 1987). Because the amplification of this integrated prophage is normally toxic, it is very difficult to obtain and propagate a host cell strain carrying the amplified integrated DNA. This then requires that the gene be amplified each instance that protein production is desired.
Diederich et al. ((1992) "New plasmid vectors for integration into the 1 attachment site attB of the Escherichia coli chromosome", Plasmid 28:14-24) also disclose a system for introducing a gene onto the chromosome of a bacterial host cell. This system utilizes a set of multicopy plasmid vectors which can be integrated into a bacterial chromosome via a phage lambda attachment site. A DNA sequence encoding a promoter operably linked to a gene of interest is cloned into one of the described multicopy number plasmid vectors, the plasmid's origin of replication is removed by restriction enzymes, and the resulting DNA is recircularized and transferred to a host cell, where it integrates into the chromosome.
These new gene transfer systems suffer from the same defect as earlier systems. Both USP 5,395,763 (Weinberg et al.) and Diederich et al. require that the gene of interest be cloned into a multicopy number plasmid while in an operable configuration during the construction of the transfer DNA. The configuration of this multicopy number plasmid makes expression of toxic foreign genes difficult, if not impossible, because the (toxic) gene of interest will be expressed as the multicopy number plasmid is propagated.
Accordingly, there is a need for a method of producing heterologous proteins which can produce large amounts of protein and which minimizes any toxic effect of the heterologous protein to host cells during construction of the producing strain. Applicants have shown surprisingly high protein accumulation (approximately 20% of total cell protein) from expression of low (approximately two) copies of the gene encoding the heterologous protein as shown in Example 2.